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The Power of Paired-Tag: Joint Profiling of Epigenetics and Gene Expression in Single Cells (Part 2)

The Applications of Single-Cell Analysis

The first in a series of articles from Epigenome Technologies described "Paired-Tag" (parallel analysis of individual cells for RNA expression and DNA from targeted tagmentation by sequencing) as an exciting means to jointly profile histone modifications and gene expression at single-cell resolution. This advance was first developed by a team guided by Bing Ren at the Ludwig Institute for Cancer Research/University of California San Diego; now, Epigenome Technologies provides optimized Paired-Tag kits and services to researchers in the epigenetics field under an exclusive license.

Researchers have linked the extensive dysregulation of gene expression to disease development; however, the precise mechanisms underpinning said alterations to vast numbers of genes remain somewhat unknown. Substantial evidence suggests that epigenetic factors play a significant, potentially targetable, role in developing and maintaining disease-associated gene expression profiles; now, Paired-Tag provides an optimized means to fully understand the epigenetic underpinnings of normal physiology and disease.

This article series reports on the vast potential of Epigenome Technologies' single-cell profiling technology by discussing the Nature Methods and Nature Structural & Molecular Biology articles that helped to bring the only commercially available technology for joint profiling of histone modifications and gene expression in single cells to the market and then describing the most recent exciting applications. Stay tuned for an upcoming article in this series that also describes a recent application of Paired-Tag to support the single-cell epigenomic and transcriptomic analysis of the Alzheimer's disease-affected human brain, which affords detailed insights into disease-associated molecular mechanisms.

Based on a Nature Methods study, a previous article described the application of Paired-Tag to mouse cells and provided the first report of combined histone modification and transcriptomic maps; now, this follow-up article on the same study describes the integrated analysis of these maps to identify genes subject to divergent epigenetic regulatory mechanisms.

Can we move forward and use such information to identify the epigenetic mechanisms underpinning disease states and identify potential exciting therapeutic targets?

Demonstrating the Power of Paired-Tag in profiling Epigenetics

Paired-Tag Integrates Histone Modification and Gene Expression Profiles at Gene Promoters in Mouse Brain Cells
  • Investigating the relationship between histone modification and cell-type-specific gene expression profiles integrated Paired-Tag signals at annotated gene promoters in each mouse brain cell type

    • This categorized gene promoters into seven groups possessing distinct histone modification combinations

    • Class I promoters associated with H3K9me3, Class II-a and b groups associated with H3K27me3, while Class III a-d groups possessed variable levels of H3K4me1 and H3K27ac

  • Gene Ontology analysis revealed distinct functional categories of genes within each group

    • Class I genes displayed strong enrichment for sensory-related pathways, Class II-a genes for developmental processes, while Class III-b genes displayed expression in all neuron types

  • Data integration supported the pseudotime analysis of oligodendrocyte lineage differentiation via transcriptomic profiles and the promoter histone modification states assigned to differentially expressed genes

    • This analysis supports the ability of Paired-Tag to evaluate epigenetic regulatory programs during development

  • The application of this Paired-Tag-supported analytical approach to disease-affected cells could enable the segregation of differentially expressed genes into sub-classes dependent on the epigenetic mechanisms involved

    • This strategy could prioritize targets for epigenetic interventions such as polycomb, SWI/SNF complex, or histone deacetylase components

Paired-Tag Analysis Describes Distal Cis-Regulatory Elements in Mouse Brain Cells
  • Characterizing candidate cis-regulatory elements (cCREs) evaluated histone modification profiles across brain cell types

    • Two cCRE groups possessed H3K9me3 in all cell types (Class eI-a) or selectively in neuronal cells (eI-b) and generally resided in intergenic regions distant from CpG islands

    • Two cCRE groups possessed H3K27me3 in all neuronal cells (Class eII-a) or a more restricted manner (eII-b), with the latter group only significantly enriched at CpG islands

    • Four cCRE groups (Class eIII-a to d) possessed variable levels of H3K4me1 and H3K27ac in different cell clusters

      • Class eIII-a cCREs with invariable H3K4me1/H3K27ac resided in genic regions and displayed enrichment at CpG islands compared to other eIII cCREs

      • Class eIII-d cCREs with elevated H3K27ac levels comprised the most prominent group

  • The analysis of matched normal and diseased cell samples by applying Paired-Tag could allow the identification of epigenetically dysregulated cCREs, which could identify both the factors implicated in disease-associated chromatin remodeling activity and disease-relevant transcription factors via an analysis of enriched motifs

Paired-Tag Analysis Links Distal Candidate Cis-Regulatory Elements to Target Genes in Mouse Brain Cells
  • H3K4me1 co-occupancy of cCREs and transcription start sites combined with correlations made between target gene expression and cCRE histone modification profiles connected active/repressive cCREs to putative target genes

    • 32,252 cCRE-gene pairs possessed H3K27ac at cCREs positively correlated with gene expression

    • 15,199 cCRE-gene pairs possessed H3K27me3 at cCREs negatively correlated with gene expression

    • Candidate CREs of "inactive pairs" displayed more significant enrichment in intergenic regions

  • A significant fraction of H3K27ac- and H3K27me3-associated cCRE-gene pairs overlapped, with cCREs tending to be Class eII-b cCREs and involving target genes enriched for development processes, which agrees with the recently reported transition between PRC2-associated silencers and active enhancers during differentiation

  • A comparison of cCRE and putative target gene categories revealed that they tended to fall into similar groups

    • As an example, target genes of Class eII-a and -b cCREs displayed a strong enrichment in promoters of Class II-a and II-b genes, which themselves associated with development processes

  • A comparison of histone modification profiles of cCREs and putative target gene promoters revealed high concordance for H3K27ac levels at active pairs and high concordance for H3K27me3 levels for inactive pairs

  • Applying this approach to diseased cells thanks to Paired-Tag could directly link cCREs to driver genes, thereby providing potential targets for gene-editing or epigenetic therapies

Bring The Power of Paired-Tag to Your Research

Jointly profile transcriptomics and epigenetics with Droplet Paired-Tag: High UMI coverage for both modalities

Paired-Tag represents an exciting commercially available means to jointly profile histone modifications and gene expression at the single-cell resolution and detect histone modifications and RNA transcripts in individual nuclei with an efficiency comparable to single-nucleus RNA-seq/ChIP–seq assays. Overall, applying Paired-Tag technology may enable quantum leaps forward in our understanding of development and significantly improve disease management.

Here, the analysis of combined histone modification and transcriptomic maps in single mouse brain cells revealed how Paired-Tag could identify genes subject to divergent epigenetic regulatory mechanisms in mouse brain cells. An upcoming article based on a Nature Structural & Molecular Biology article continues our introduction to Paired-Tag technology and describes a faster and more accessible droplet-based Paired-Tag protocol.

by Stuart P. Atkinson

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